The identification and evaluation of microRNAs is one of the fastest growing fields in biology and medicine. MicroRNAs are considered to be key elements in maintaining normal cell physiology as microRNAs appear to be central regulators of gene expression that are critical for intra- and extra-cellular events. Thus, either the loss of specific microRNAs or the overexpression of specific microRNAs can lead to abnormal cell processes that are the underlying basis for disease. Inhibition of an aberrant microRNA with an antagonist, or the supplementation with a microRNA mimetic, can restore the balance between cellular communication pathways. Therapeutics approaches using nucleic acid compounds containing a CRN can directly and efficiently alter the function of microRNAs.
Nucleic acid compounds containing a conformationally restricted nucleomonomer or CRN can be used as microRNA antagonists. The therapeutic benefit of inhibiting a specific microRNA is the de-repression of downstream targets controlled by the microRNA. De-repression allows the downstream targets to express proteins which the microRNA typically down regulates. Thus, microRNA inhibition is one of the few therapeutic approaches that permits “upregulation” of specific gene targets.
RNA interference or RNAi refers to the cellular process of sequence specific, post-transcriptional gene silencing mediated by small inhibitory nucleic acid molecules. An RNAi active molecule can be a double-stranded RNA or dsRNA that has a portion that is homologous to a portion of a targeted messenger RNA. A long dsRNA may be processed by Dicer enzyme into a short interfering RNA or siRNA having from 21 to 23 nucleotides with double-stranded regions of about 19 base pairs and a two nucleotide, generally, overhang at each 3′-end. An siRNA can interact with an RNA-induced silencing complex or RISC complex which cleaves the passenger or sense strand of the siRNA. The guide or antisense strand of the siRNA can bind a complementary target mRNA, which is then cleaved by the RISC to cause gene silencing.
Nucleic acid compounds containing a CRN can be used to increase the affinity of a single-stranded oligonucleotide to its intended target, whether it is a messenger RNA or microRNA.
Nucleic acid compounds containing a CRN can be used to silence gene targets through either RNA interference or translational blocking, or via a microRNA mimetic. They can be used to up-regulate gene targets via a microRNA antagonist.
What is needed are efficient methods for preparing nucleomonomers for nucleic acid compounds in high yield at multi-gram scale for therapeutic modalities useful for treating or preventing diseases or disorders by up- or down-regulating the expression of genes and other nucleic acid based regulatory systems in a cell.
A need therefore exists for methods for preparing nucleomonomers for nucleic acid compounds having enhanced stability that are useful in various therapeutic modalities involving microRNA, siRNA, and/or antisense RNA.